Current antiretroviral combination therapy is extremely effective at suppressing HIV-1 viremia below to the limit of detection of standard clinical assays, however it is unable to fully eradicate HIV-1. As a consequence, once treatment is stopped, HIV-1 replication can rapidly rebound from the latent reservoir, and typically reaches pre-treatment levels of HIV-1 replication within a short period of time. Thus, the understanding of mechanisms contributing to HIV-1 persistence despite ART, and the development of therapeutic strategies that target persistent virus, represent one of the highest priorities in current HIV-1 research. In this application, we hypothesize that in samples from long- term treated individuals, infected long-lived CD4 T cells will preferentially express HIV RNA-TAR, which will confer to the cells a survival advantage. Thus, here we propose to deeply characterize single cells exclusively expressing HIV RNA-TAR in CD4 T subsets from ART-treated patients. In addition, HIV integration sites sequencing will be used to determine the long-term persistence and the clonal expansion capabilities of cells expressing HIV RNA-TAR during prolonged ART. If successful, these investigations may lead to the identification of the exclusive signatures of latently infected cells in vivo, re-directing current efforts in design clinical straegies aimed at cure HIV, thus addressing one of the highest-priority issues in current HIV-1 research.